Epicardial formation is necessary for normal myocardial morphogenesis. Here, we show that differentiating hiPSC-derived lateral plate mesoderm with BMP4, RA and VEGF (BVR) can generate a premature form of epicardial cells (termed pre-epicardial cells, PECs) expressing WT1, TBX18, SEMA3D, and SCX within 7 days. BVR stimulation after Wnt inhibition of LPM demonstrates co-differentiation and spatial organization of PECs and cardiomyocytes (CMs) in a single 2D culture. Co-culture consolidates CMs into dense aggregates, which then form a connected beating syncytium with enhanced contractility and calcium handling; while PECs become more mature with significant upregulation of UPK1B, ITGA4, and ALDH1A2 expressions. Our study also demonstrates that PECs secrete IGF2 and stimulate CM proliferation in co-culture. Three-dimensional PEC-CM spheroid co-cultures form outer smooth muscle cell layers on cardiac micro-tissues with organized internal luminal structures. These characteristics suggest PECs could play a key role in enhancing tissue organization within engineered cardiac constructs in vitro.
Decellularized native extracellular matrix (ECM) biomaterials are widely used in tissue engineering and have reached clinical application as biomesh implants. To enhance their regenerative properties and postimplantation performance, ECM biomaterials could be functionalized via immobilization of bioactive molecules. To facilitate ECM functionalization, we developed a metabolic glycan labeling approach using physiologic pathways to covalently incorporate click-reactive azide ligands into the native ECM of a wide variety of rodent tissues and organs in vivo, and into the ECM of isolated rodent and porcine lungs cultured ex vivo. The incorporated azides within the ECM were preserved after decellularization and served as chemoselective ligands for subsequent bioconjugation via click chemistry. As proof of principle, we generated alkyne-modified heparin, immobilized it onto azide-incorporated acellular lungs, and demonstrated its bioactivity by Antithrombin III immobilization and Factor Xa inhibition. The herein reported metabolic glycan labeling approach represents a novel platform technology for manufacturing click-reactive native ECM biomaterials, thereby enabling efficient and chemoselective functionalization of these materials to facilitate tissue regeneration and repair.